<p>The description was made of the karyotype of <em>Peromyscus gratus</em> (Cricetidae) in the municipality of Tecamachalco, Puebla. Performing the technique of bone marrow extraction, in the obtention of chromosome, G bands was used Trypsin and for C bands a Barium Hydroxide solution was employed. <em>P</em>. <em>gratus</em> presents a diploid number of 2n = 48 and a fundamental number of NF = 54, the autosomes correspond to one metacentric, one submetacentric, two subtelocentric and 19 pairs telocentric from large to small. The sex chromosome X was subtelocentric and the Y was submetacentric unlike other populations of the species. The chromosomal banding pattern G was obtained. As for chromosome C banding, it was found that the constitutive heterochromatin was in the centromeric regions of the chromosomes.</p>